nutrients

Article
Impact of Pre-Pregnancy BMI on B Vitamin and
Inflammatory Status in Early Pregnancy:
An Observational Cohort Study
Anne-Lise Bjørke-Monsen 1, *, Arve Ulvik 2 , Roy M. Nilsen 3 , Øivind Midttun 2 ,
Christine Roth 4,5 , Per Magnus 4 , Camilla Stoltenberg 4,6 , Stein Emil Vollset 4,6 ,
Ted Reichborn-Kjennerud 4,7 and Per Magne Ueland 1,8
1 Laboratory of Clinical Biochemistry, Haukeland University Hospital, 5021 Bergen, Norway;
per.ueland@ikb.uib.no
2 Bevital AS, 5021 Bergen, Norway; Arve.Ulvik@uib.no (A.U.); oivind.midttun@bevital.no (Ø.M.)
3 Centre for Clinical Research, Haukeland University Hospital, 5021 Bergen, Norway; Roy.Nilsen@uib.no
4 The Norwegian Institute of Public Health, 0403 Oslo, Norway; Christine.Roth@fhi.no (C.R.);
Per.Magnus@fhi.no (P.M.); camilla.stoltenberg@fhi.no (C.S.); SteinEmil.Vollset@fhi.no (S.E.V.);
Ted.Reichborn-Kjennerud@fhi.no (T.R.-K.)
5 Nic Waals Institute, Lovisenberg Hospital, Oslo 0456, Norway
6 Department of Public Health and Primary Health Care, University of Bergen, 5007 Bergen, Norway
7 Institute of Clinical Medicine, University of Oslo, 0313 Oslo, Norway
8 Department of Clinical Science, University of Bergen, 5007 Bergen, Norway
* Correspondance: almo@helse-bergen.no; Tel.: +47-559-730-87; Fax: +47-559-731-15

Received: 8 September 2016; Accepted: 28 November 2016; Published: 30 November 2016

Abstract: Maternal nutrition and inflammation have been suggested as mediators in the development
of various adverse pregnancy outcomes associated with maternal obesity. We have investigated
the relation between pre-pregnancy BMI, B vitamin status, and inflammatory markers in a group
of healthy pregnant women. Cobalamin, folate, pyridoxal 50 -phosphate, and riboflavin; and the
metabolic markers homocysteine, methylmalonic acid, and 3-hydroxykynurenine/xanthurenic acid
ratio (HK/XA); and markers of cellular inflammation, neopterin and kynurenine/tryptophan ratio
(KTR) were determined in pregnancy week 18 and related to pre-pregnancy body mass index (BMI),
in 2797 women from the Norwegian Mother and Child Cohort Study (MoBa). Pre-pregnancy BMI
was inversely related to folate, cobalamin, pyridoxal 50 -phosphate (PLP), and riboflavin (p < 0.001),
and associated with increased neopterin and KTR levels (p < 0.001). Inflammation seemed to be an
independent predictor of low vitamin B6 status, as verified by low PLP and high HK/XA ratio. A high
pre-pregnancy BMI is a risk factor for low B vitamin status and increased cellular inflammation. As an
optimal micronutrient status is vital for normal fetal development, the observed lower B vitamin
levels may contribute to adverse pregnancy outcomes associated with maternal obesity and B vitamin
status should be assessed in women with high BMI before they get pregnant.

Keywords: pregnancy; obesity; pre-pregnancy BMI; B vitamins; inflammation

1. Introduction
There is an increasing prevalence of obesity in most parts of the world, also affecting women
of childbearing age [1]. Pre-pregnancy obesity is associated with an increased risk of adverse
pregnancy outcomes for both mother and child, including subfertility, miscarriage, gestational diabetes,
gestational hypertension, preeclampsia, macrosomia, preterm birth, congenital anomalies, and fetal
death [2,3]. Epidemiological studies have revealed strong links between nutritional excess during
pregnancy and later development of metabolic disease, such as type 2 diabetes and obesity, in adult

Nutrients 2016, 8, 776; doi:10.3390/nu8120776 www.mdpi.com/journal/nutrients

Nutrients 2016, 8, 776 2 of 15

life [4]. Maternal metabolic, nutritional, and inflammatory factors have been suggested as mediators
in the development of the various negative pregnancy outcomes associated with maternal obesity.
A higher body mass index (BMI) has been associated with an adverse nutritional status in both
non-pregnant adults [5–7] and children [8,9]. An inverse relation between pre-pregnancy BMI and
several micronutrients like folate, vitamin D, carotenoids, zinc, and essential fatty acids have been
reported in pregnant women, and negative pregnancy outcomes related to obesity might be related to
impaired micronutrient status [10–13]. An adequate B vitamin status during pregnancy is important
for maternal health and normal fetal development [14]. Deficiencies of folate, cobalamin, riboflavin, or
vitamin B6 are associated with an increased risk of placental abruption, still-births, low birth weight,
preterm deliveries, preeclampsia, as well as fetal malformations [14].
Obesity in itself results in an inflammatory state in metabolic tissues [15], and higher levels of the
inflammatory markers C-reactive protein, neopterin, and kynurenine/tryptophan ratio (KTR) are seen
in overweight and obese adults [16,17]. Inflammation has been associated with low circulating levels
of several micronutrients [18], such as the active form of vitamin B6 , pyridoxal 50 -phosphate (PLP) [19],
and vitamin D [20].
The objective of the study was to investigate whether BMI and inflammation are independent
determinants of B vitamin status, we investigated the association of pre-pregnancy BMI with plasma B
vitamin status and inflammatory markers in pregnancy week 18 in 2797 women from the Norwegian
Mother and Child Cohort Study (MoBa).

2. Experimental Section

2.1. Study Population

This study is based on a subsample of 2825 women included in the Norwegian Mother and
Child Cohort Study (MoBa), a long-term, prospective study conducted by the Norwegian Institute
of Public Health and including more than 100,000 Norwegian pregnant women and their infants
during 1999–2008 [21]. The women included in this sub-study of MoBa gave a singleton birth between
July 2002 and December 2003, and returned a baseline questionnaire including data on height and
pre-pregnancy weight and donated a blood sample in pregnancy week 18, and were registered in the
Medical Birth Registry of Norway. A detailed description of this sub population and the sampling
procedures has been published [22].

2.2. Ethics
Written informed consent was obtained from each participant, and the study was approved
by the Regional Committee for Medical Research Ethics Sør-Øst A, 19/10-2011 (permission number
2009/2593a) and the Norwegian Data Inspectorate. All questions used in the MoBa can be found
online at www.fhi.no/morogbarn.

2.3. Blood Sampling and Laboratory Analyses

Maternal non-fasting blood samples were collected in ethylenediamine tetraacetic acid (EDTA)
tubes in median pregnancy week 18. The samples were centrifuged within 30 min after collection and
stored at 4 ◦ C until shipped overnight to the Norwegian Institute of Public Health in Oslo. On the day
of receipt (usually within 1–2 days), plasma was aliquoted into polypropylene microtiter plates and
stored at −80 ◦ C until analyses.
Plasma folate was determined by a Lactobacillus casei microbiological assay [23] and plasma
cobalamin (vitamin B12) by a Lactobacillus leichmannii microbiological assay [24]. Concurrent intake
of antibiotics may interfere with microbiological assays and cause falsely reduced plasma levels of
the vitamins [25], and samples with plasma folate levels <2.33 nmol/L were excluded (n = 28, i.e., the
lower one percentile), leaving a total of 2797 samples to be included in the study.
Nutrients 2016, 8, 776 3 of 15

Plasma levels of total homocysteine (tHcy), a marker of folate and cobalamin status, and
methylmalonic acid (MMA), a marker of cobalamin status, were assayed using a GC-MS method based
on methylchloroformate derivatization [26]. Plasma levels of riboflavin (vitamin B2); pyridoxal
5’-phosphate (PLP; vitamin B6); the ratio between 3-hydroxykynurenine and xanthurenic acid
(HK/XA), a marker of vitamin B6 status [27]; and two markers of interferon-gamma mediated
cellular immune activation, neopterin [28] and KTR (Kynurenine/Tryptophan × 1000)—the
latter reflecting indoleamine-2,3-dioxygenase (IDO) activation [29]—were analyzed using a liquid
chromatography-tandem mass spectrometry assay [30]. All biomarkers were measured at the
laboratory of Bevital AS (www.bevital.no).

2.4. Covariates
Data on maternal age at delivery, marital status, and parity were obtained from the Medical
Birth Registry of Norway. Data on maternal education, smoking habits, pre-pregnancy BMI, weight
increase to pregnancy week 18, and use of supplements were obtained from the self-reported baseline
cohort questionnaire. Pre-pregnancy BMI (kg/m2 ) was coded as <18.5, 18.5–24.9, 25.0–29.9, 30.0–34.9,
and ≥35.0. Use of supplements was coded as non-user, use of folic acid or other supplement, and use
of folic acid plus other supplement, based on reported intake from four weeks prior to conception up
to pregnancy week 18. A B vitamin summary score was calculated as a sum of quintiles for folate,
cobalamin, PLP, and riboflavin.

2.5. Statistical Analyses

Values are presented as means with standard deviation (SD) or medians with the interquartile
range. Differences between groups were examined by ANOVA, Mann-Whitney U test and the Kruskal
Wallis test. Differences in categorical variables were assessed with the Chi-square test. Multiple-linear
regression models were used to assess the relation between pre-pregnancy BMI, use of supplements,
parity, age, and inflammation with plasma B vitamin status obtained around pregnancy week 18.
The odds ratio (ORs) for having a low B vitamin status (quintile 1) according to maternal factors,
including the inflammation marker neopterin, was assessed by logistic regression.
To explore non-linearity, we modeled pre-pregnancy BMI versus B vitamins and metabolic
markers, using one-dimensional smoothing splines in Generalized Additive Models (GAM), in a model
adjusted for adjusted for use of supplements, maternal age, parity, and neopterin.
GAMs were computed using the package mgcv (version 1.4–1) in R (The R Foundation for
Statistical Computing, version 2.8.1, Vienna, Austria) [31], and the SPSS/PASW statistical program
version 22 (IBM Corp, Armonk, NY, USA).the company, the city, the country) was used for all other
statistical analyses. Two-sided p-values < 0.05 were considered statistically significant.

3. Results

3.1. Characteristics of the Study Population According to Pre-Pregnancy BMI

Demographic characteristics according to pre-pregnancy BMI are presented in Table 1. Fifty-eight
percent of the women were multipara, and had a mean number of 1.5 (SD 0.7) former children, with
no difference according to BMI group (p = 0.14). The majority of the women were healthy, a low
percentage (0.7%) had chronic hypertension and 2% had had IVF treatment. The use of multivitamin
supplements differed according to BMI group, with an increasing trend for not using any supplements
with higher BMI (p < 0.001). Regular use of alcohol was rare and 96% reported drinking alcohol never
or less than once a month after becoming pregnant. A total of 231 (8%) women reported daily smoking
with a mean number of seven (SD five) cigarettes per day, with no significant difference in number
between the smokers in the various BMI groups (p = 0.80). There was an inverse relation between
pre-pregnancy BMI and weight gain to pregnancy week 18 (r = −0.24, p < 0.001) and to term (r = −0.16,
p < 0.001).
Nutrients 2016, 8, 776 4 of 15

Table 1. Maternal characteristics according to maternal pre-pregnancy BMI, n = 2797.

Pre-pregnancy BMI, Categories

<18.5 18.5–24.9 25.0–29.9 30.0–34.9 ≥35.0 p Value
n = 80 (3%) n = 1827 (65%) n = 587 (21%) n = 213 (8%) n = 90 (3%)
Primipara, n (%) 47 (59%) 785 (43%) 231 (39%) 86 (40%) 26 (29%) 0.001 b
Age, years, mean (SD) 27.8 (4.9) 29.9 (4.5) 29.9 (4.6) 29.8 (4.6) 29.5 (4.8) 0.003 a
Education
Primary school, n (%) 9 (12%) 39 (2%) 15 (3%) 11 (5%) 6 (7%)
Secondary school, n (%) 30 (41%) 623 (36%) 273 (48%) 97 (47%) 48 (55%) <0.001 b
University or college, n (%) 35 (47%) 1077 (62%) 286 (50%) 98 (48%) 34 (39%)
Use of supplements anytime from four weeks
63 (79%) 1523 (83%) 470 (79%) 156 (72%) 61 (68%) <0.001 b
before pregnancy up to pregnancy week 18
Use of alcohol (≥1/month), n (%) 5 (0.2%) 83 (3%) 15 (0.6%) 1 (0%) 3 (0.1%) 0.009 b
Daily smoking, n (%) 13 (16%) 140 (8%) 47 (8%) 22 (10%) 9 (10%) 0.04 b
Weight increase, kg, mean (SD), (% of
pre-pregnancy weight)
to pregnancy week 18 4.5 (3.1) (9%) 3.3 (2.9) (5%) 2.4 (3.2) (3%) 1.1 (3.5) (1%) −0.1 (4.3) (−0.1%) <0.001 a <0.001 a
to birth 17.3 (6.3) (35%) 15.5 (5.9) (25%) 14.8 (6.7) (20%) 11.5 (7.3) (13%) 8.7 (8.9) (8%) <0.001 a <0.001 a
Birth weight, g, mean (SD) 3343 (635) 3575 (588) 3727 (600) 3696 (648) 3758 (755) <0.001 a
a ANOVA: A one-way variance analysis; b Pearson’s Chi Square test. BMI: body mass index; SD: the standard deviation.
Nutrients 2016, 8,
Nutrients 2016, 8, x776
FOR PEER REVIEW 55 of
of 15
15

Women in the highest pre-pregnancy BMI group (BMI ≥ 35) put on less weight during
pregnancy,
Womenthey were
in the morepre-pregnancy
highest likely to be multipara,
BMI group not(BMI
use ≥micronutrient
35) put on lesssupplements,
weight during andpregnancy,
to have a
lower educational level (p < 0.01). The lowest mean birth weight was seen in women witha alower
they were more likely to be multipara, not use micronutrient supplements, and to have pre-
pregnancy
educationalBMI level<18.5
(p < (Table 1). lowest mean birth weight was seen in women with a pre-pregnancy
0.01). The
BMI <18.5 (Table 1).
3.2. Plasma B Vitamin Status According to Pre-Pregnancy BMI
3.2. Plasma B Vitamin Status According to Pre-Pregnancy BMI
There was a trend towards lower levels of B vitamins and higher levels of the metabolic markers
with There
higherwaspre-pregnancy
a trend towardsBMIlower
(Table 2, Figure
levels 1). The best
of B vitamins B vitamin
and higher status
levels of thewas seen in markers
metabolic women
with a normal pre-pregnancy BMI (18.5–24.9), as they had the highest levels of folate, PLP, with
with higher pre-pregnancy BMI (Table 2, Figure 1). The best B vitamin status was seen in women and
riboflavin with the lowest
a normal pre-pregnancy BMItHcy and HK/XA
(18.5–24.9), ratio
as they had(Table 2). Cobalamin
the highest levels ofdecreased
folate, PLP,with
andincreasing
riboflavin
BMI, the
with butlowest
this was tHcynotand
reflected
HK/XA in ratio
the MMA(Tablelevels, which were
2). Cobalamin inversely
decreased withrelated to pre-pregnancy
increasing BMI, but this
BMI.
was not reflected in the MMA levels, which were inversely related to pre-pregnancy BMI.
The
The circulating
circulatingB-vitamins
B-vitaminswere were inversely
inversely related to their
related functional
to their markers,
functional as expected.
markers, tHcy
as expected.
tHcy was inversely related to folate (r = −
was inversely related to folate (r = −0.48, p < 0.001), less to cobalamin (r = −0.24, p < 0.001), PLP
0.48, p < 0.001), less to cobalamin (r = − 0.24, p < 0.001), PLP
(r = −
(r = −0.16, p < 0.001), and riboflavin (r = −0.15, p < 0.001). MMA was inversely related to cobalamin
0.16, p < 0.001), and riboflavin (r = − 0.15, p < 0.001). MMA was inversely related to cobalamin
(r −0.17,pp<<0.001)
(r == −0.17, 0.001)andandHK/XA
HK/XA toto
PLPPLP (r (r = −0.29,
= −0.29, p <p0.001). AllAll
< 0.001). B vitamins
B vitamins were positively
were positivelyrelated to
related
each other (r > 0.12, p < 0.001).
to each other (r > 0.12, p < 0.001).

Figure 1. The association of pre-pregnancy BMI with cobalamin, folate, PLP, riboflavin, tHcy, MMA,
Figure 1. The association of pre-pregnancy BMI with cobalamin, folate, PLP, riboflavin, tHcy, MMA,
and HK/XA by generalized additive models (GAM), adjusted for use of multisupplements, maternal
and HK/XA by generalized additive models (GAM), adjusted for use of multisupplements, maternal
age, parity, and neopterin. The solid line shows the fitted model and the shaded areas indicate 95%
age, parity, and neopterin. The solid line shows the fitted model and the shaded areas indicate 95%
CIs. PLP, pyridoxal 5′phosphate; tHcy, total homocysteine; MMA, methylmalonic acid, HK/XA, 3-
CIs. PLP, pyridoxal 50 phosphate; tHcy, total homocysteine; MMA, methylmalonic acid, HK/XA,
hydroxykynurenine/xanthurenic acid ratio.
3-hydroxykynurenine/xanthurenic acid ratio.
Nutrients 2016, 8, 776 6 of 15

Table 2. Maternal plasma levels of vitamins, metabolic and inflammation markers in pregnancy week 18 according to pre-pregnancy BMI, n = 2797.

Pre-pregnancy BMI; Categories

<18.5 18.5–24.9 25.0–29.9 30.0–34.9 ≥35.0 p Value
n = 80 n = 1827 n = 587 n = 213 n = 90
Plasma folate, nmol/L a 8.8 (6.0–15.7) 9.2 (6.3–16.0) 8.1 (5.7–12.8) 7.3 (5.1–12.2) 7.3 (5.0–12.1) <0.001
Plasma cobalamin, pmol/L a 328 (243–383) 314 (254–386) 301 (240–363) 271 (220–338) 268 (204–317) <0.001
Plasma PLP, nmol/L a 27.0 (21.4–39.7) 28.3 (21.8–40.7) 26.6 (20.6–35.6) 23.8 (17.4–33.7) 21.5 (17.0–30.1) <0.001
Plasma riboflavin, nmol/L a 7.8 (4.8–14.4) 8.1 (5.6–13.2) 7.8 (5.2–13.9) 7.2 (5.2–11.0) 6.9 (4.4–10.1) 0.004
B vitamin status a,b 12.2 (3.7) 12.5 (3.6) 11.7 (3.7) 10.6 (3.7) 10.1 (3.4) <0.001
Plasma tHcy, µmol/L a 5.00 (4.22–5.92) 4.91 (4.27–5.79) 4.94 (4.23–5.78) 5.04 (4.44–6.21) 5.33 (4.43–6.13) 0.03
Plasma MMA, µmol/L a 0.13 (0.11–0.16) 0.13 (0.11–0.16) 0.13 (0.10–0.16) 0.12 (0.10–0.15) 0.12 (0.10–0.15) 0.009
Plasma HK/XA a 1.6 (1.2–2.1) 1.4 (1.0–2.0) 1.5 (1.1–2.1) 1.7 (1.2–2.2) 1.8 (1.2–2.4) <0.001
Plasma neopterine, µmol/L a 6.7 (6.1–8.1) 7.0 (6.1–8.1) 7.3 (6.3–8.6) 7.9 (7.0–9.1) 8.5 (7.31–9.9) <0.001
KTR, nmol/µmol a 18.1 (16.0–21.5) 18.3 (16.2–20.6) 18.8 (16.8–21.2) 19.6 (17.5–22.8) 21.2 (18.5–24.0) <0.001
aMedian (IQR), by Kruskal Wallis test; b B vitamin status; summary score based on added quintiles of the 4 B vitamins, Mean (SD), by ANOVA; tHcy, total homocysteine;
MMA, methylmalonic acid; HK/XA: 3-hydroxykynurenine/xanthurenic acid; KTR, kynurenine/tryptophan ratio.
Nutrients 2016, 8, 776 7 of 15
Nutrients 2016, 8, x FOR PEER REVIEW 7 of 15

3.3. Inflammation
3.3. Inflammation Markers
Markers According
Accordingto
toPre-Pregnancy
Pre-PregnancyBMI
BMI
There was
There was aa positive
positive correlation
correlation between
between pre-pregnancy
pre-pregnancy BMI BMI and
and the
the inflammation
inflammation markers
markers
neopterin 0.18,p p< <0.001)
neopterin(r(r==0.18, 0.001)and
and KTR
KTR (r 0.13,
(r = p <p0.001),
= 0.13, < 0.001), as demonstrated
as demonstrated in Figure
in Figure 2. Neopterin
2. Neopterin and
and KTR
KTR werewere strongly
strongly correlated
correlated to other
to each each other (r = 0.48,
(r = 0.48, p < 0.001).
p < 0.001).

Figure 2. The association of pre-pregnancy BMI with neopterin and KTR by Generalized additive
Figure 2. The association of pre-pregnancy BMI with neopterin and KTR by Generalized additive
models (GAM), adjusted for use of multisupplements, maternal age, and parity. The solid line shows
models (GAM), adjusted for use of multisupplements, maternal age, and parity. The solid line shows
the fitted model and the shaded areas indicate 95% CIs. KTR, kynurenine/tryptophan ratio.
the fitted model and the shaded areas indicate 95% CIs. KTR, kynurenine/tryptophan ratio.

3.4. Maternal Factors and Inflammation Markers as Determinants of Plasma B Vitamin Status in Pregnancy
3.4.
WeekMaternal
18 Factors and Inflammation Markers as Determinants of Plasma B Vitamin Status in Pregnancy
Week 18
Use of supplements was the most consistent and strongest determinant for maternal B vitamin
Use of supplements was the most consistent and strongest determinant for maternal B vitamin
status, with significant influence on folate, cobalamin, PLP, riboflavin, tHcy, and HK/XA, in a
status, with significant influence on folate, cobalamin, PLP, riboflavin, tHcy, and HK/XA, in a multiple
multiple linear regression model, which additionally included pre-pregnancy BMI, age, parity, and
linear regression model, which additionally included pre-pregnancy BMI, age, parity, and neopterin
neopterin (Table 3). In this model, pre-pregnancy BMI was a significant determinant of all vitamin
(Table 3). In this model, pre-pregnancy BMI was a significant determinant of all vitamin markers except
markers except for riboflavin and tHcy, whereas parity was strongly negatively correlated to folate
for riboflavin and tHcy, whereas parity was strongly negatively correlated to folate and PLP status.
and PLP status. All parameters in the model were significantly correlated to the calculated B vitamin
All parameters in the model were significantly correlated to the calculated B vitamin summary score
summary score (Table 3). Neopterin was significantly negative related to PLP, and positively to the
(Table 3). Neopterin was significantly negative related to PLP, and positively to the metabolic markers
metabolic markers tHcy, MMA, and HK/XA. Substituting neopterin with KTR in the model did not
tHcy, MMA, and HK/XA. Substituting neopterin with KTR in the model did not essentially change
essentially change the results (data not shown). Including gestational weight gain in pregnancy week
the results (data not shown). Including gestational weight gain in pregnancy week 18 as an additional
18 as an additional factor did not change the results.
factor did not change the results.
The OR for having a low calculated B vitamin summary score (quintile 1) was increased with
The OR for having a low calculated B vitamin summary score (quintile 1) was increased with
pre-pregnancy BMI >30.0 and higher parity, and reduced with higher maternal age and use of
pre-pregnancy BMI >30.0 and higher parity, and reduced with higher maternal age and use of
supplements in a logistic regression model, which additionally included neopterin quartiles (Table
supplements in a logistic regression model, which additionally included neopterin quartiles (Table 4).
4). A high pre-pregnancy BMI was a predictor of low cobalamin and PLP levels, multiparity was a
A high pre-pregnancy BMI was a predictor of low cobalamin and PLP levels, multiparity was
predictor of low folate levels, higher maternal age was associated with better folate and riboflavin
a predictor of low folate levels, higher maternal age was associated with better folate and riboflavin
status, whereas use of supplements was associated with better status of all four B vitamins. Increased
status, whereas use of supplements was associated with better status of all four B vitamins. Increased
inflammation, evaluated by higher neopterin levels, was only a predictor of low PLP in this model.
inflammation, evaluated by higher neopterin levels, was only a predictor of low PLP in this model.
Substituting neopterin with KTR in the model did not essentially change the results.
Substituting neopterin with KTR in the model did not essentially change the results.
Nutrients 2016, 8, 776 8 of 15

Table 3. Determinants of maternal plasma B vitamin status in pregnancy week 18 by multiple linear regression, n = 2797.

Plasma Plasma Plasma

Plasma PLP, Plasma tHcy, Plasma MMA, Plasma B Vitamin
Folate, Cobalamin, Riboflavin,
nmol/L µmol/L µmol/L HK/XA Score a
nmol/L pmol/L nmol/L
B (95% CI) B (95% CI) B (95% CI) B (95% CI) B (95% CI) B (95% CI) B (95% CI) B (95% CI)
−0.59 ** −16.7 ** −2.55 ** −0.60 −0.02 −0.006 ** 0.11 * −0.47 **
Pre-pregnancy BMI c
(−0.98, −0.20) (−22.0, −11.4) (−3.72, −1.38) (−1.25, 0.06) (−0.16, 0.12) (−0.008, −0.003) (0.04, 0.18) (−0.60, −0.35)
−1.33 ** −3.6 −1.28 * −1.08 * −0.01 −0.001 −0.03 −0.34 **
Parity e
(−1.72, −0.95) (−8.8, 1.6) (−2.43, −0.12) (−1.73, −0.43) (−0.15, 0.13) (−0.004, 0.002) (−0.09, 0.04) (−0.47, −0.22)
2.69 ** 12.3 * 1.96 * 0.30 0.04 0.002 −0.12 * 0.63 **
Age d
(2.05, 3.32) (3.6, 20.9) (0.06, 3.86) (−0.77, 1.36) (−0.19, 0.27) (−0.002, 0.007) (−0.24, −0.01) (0.43, 0.83)
5.14 ** 13.0 * 8.49 ** 1.80 * −0.75 ** 0.001 −0.21 * 1.73 **
Use of supplements b
(4.35, 5.94) (2.3, 23.7) (6.12, 10.85) (0.48, 3.13) (−1.03, −0.47) (−0.004, 0.007) (−0.35, −0.07) (1.48, 1.98)
−0.12 −1.9 −1.00 * −0.08 0.10 * 0.003 ** 0.19 ** −0.12 **
Neopterin f
(−0.35, 0.11) (−5.0, 1.2) (−1.68, −0.31) (−0.47, 0.30) (0.02, 0.18) (0.001, 0.004) (0.15, 0.23) (−0.20, −0.05)
All variables were included in the model; a B vitamin score; added quintiles of folate, cobalamin, PLP and riboflavin; b No use versus use of supplements any time from four weeks
before pregnancy up to pregnancy week 18; c Pre-pregnancy BMI, categorized; <18.5, 18.5-24.9, 25.0-29.9, 30.0-34.9, ≥35.0; d Age, categorized; <25 years, 25–35 years, >35 years; e Parity,
categorized; 0, 1, 2, 3+; f Neopterin, quintiles; ≤5.84, 5.85–6.68, 6.69–7.50, 7.51–8.67, ≥8.68;* p value < 0.05; ** p value < 0.001. CI: indicates confidence interval.
Nutrients 2016, 8, 776 9 of 15

Table 4. OR for low B vitamin status according to maternal factors, by logistic regression (n = 2797).

OR (95% CI) for

Plasma Folate Plasma Cobalamin Plasma PLP Plasma Riboflavin
Independent Variables B Vitamin Score a
<5.45 nmol/L <230 pmol/L <19.26 µmol/L <4.91 µmol/L
(Quintile 1)
(Quintile 1) (Quintile 1) (Quintile 1) (Quintile 1)
Pre-pregnancy BMI (vs. category 2 18.5–24.9)
<18.5 1.0 (0.5–1.8) 1.1 (0.6–1.9) 1.0 (0.5–1.8) 1.7 (1.0–2.8) 1.1 (0.7–1.9)
25.0–29.9 1.1 (0.9–1.5) 1.3 (1.0–1.6) 1.3 (1.0–1.6) 1.3 (1.0–1.7) 1.5 (1.2–1.8)
30.0–34.9 1.5 (1.0–2.1) 2.1 (1.5–2.9) 2.1 (1.6–3.0) 1.4 (1.0–2.0) 2.7 (2.0–3.7)
≥35.0 1.7 (1.0–2.8) 2.3 (1.4–3.6) 2.2 (1.4–3.5) 1.8 (1.1–3.0) 2.8 (1.7–4.3)
p trend 0.006 <0.001 <0.001 0.001 <0.001
Parity (vs. primipara)
Para 1 1.2 (1.0–1.6) 1.1 (0.9–1.4) 1.3 (1.0–1.6) 1.3 (1.0–1.6) 1.3 (1.0–1.6)
Para 2 1.7 (1.2–2.3) 1.3 (1.0–1.7) 1.1 (0.8–1.5) 1.2 (0.9–1.6) 1.6 (1.2–2.1)
Para 3+ 3.2 (2.1–4.9) 1.6 (1.0–2.4) 1.1 (0.7–1.8) 1.4 (0.9–2.2) 2.1 (1.4–3.1)
p trend <0.001 0.02 0.36 0.11 <0.001
Maternal age (vs. ≤25 years)
25–35 0.4 (0.3–0.6) 0.7 (0.6–1.0) 0.7 (0.7–1.0) 0.7 (0.6–1.0) 0.5 (0.4–0.7)
≥35 0.3 (0.2–0.5) 0.8 (0.5–1.2) 0.8 (0.5–1.1) 0.6 (0.4–0.9) 0.4 (0.3–0.6)
p trend <0.001 0.11 0.11 0.004 <0.001
Use of supplements (vs. non-user)
User 0.2 (0.2–0.2) 0.8 (0.6–1.0) 0.5 (0.4–0.6) 0.6 (0.5–0.8) 0.3 (0.3–0.4)
p trend <0.001 0.03 <0.001 <0.001 <0.001
Neopterin (vs. ≤6.20 µmol/L, Quartile 1)
6.21–7.17 1.1 (0.8–1.4) 1.1 (0.8–1.5) 1.1 (0.8–1.5) 0.9 (0.7–1.2) 1.0 (0.8–1.3)
7.18–8.40 1.3 (1.0–1.8) 1.0 (0.8–1.4) 1.1 (0.8–1.4) 0.9 (0.7–1.1) 1.1 (0.9–1.4)
≥8.41 1.2 (0.9–1.6) 1.2 (0.9–1.6) 1.7 (1.3–2.2) 0.9 (0.7–1.2) 1.2 (1.0–1.6)
p trend 0.11 0.24 <0.001 0.38 0.05
All variables were included in the model; a B vitamin score; added quintiles of folate, cobalamin, PLP and riboflavin. OR: Odds ratio.
Nutrients 2016, 8, 776 10 of 15

The relations between pre-pregnancy BMI and B vitamins and metabolic markers, visualized
by GAM in a model which additionally corrected for use of supplements, maternal age, parity, and
neopterin, are shown in Figure 1. The association curves for folate, cobalamin, PLP, and MMA show a
linear negative relation to pre-pregnancy BMI, while the relation between pre-pregnancy BMI and the
other biomarkers are inverse U-shaped (riboflavin), null (tHcy), or inverse in the lower range (HK/XA).
The association curves between pre-pregnancy BMI and the inflammation markers, neopterin
and KTR, visualized by GAM in a model which also corrected for use of supplements, maternal age,
and parity, show a strong linear relation through the whole range of BMI values (Figure 2).

4. Discussion

4.1. Main Findings

In this study of healthy women in pregnancy week 18, use of multi supplements was the strongest
maternal determinant for B vitamin status. Pre-pregnancy BMI was inversely related to folate,
cobalamin, PLP, and riboflavin levels, and associated with increased markers of cellular immune
activation. Inflammation seemed to be an independent predictor of low PLP levels.

4.2. Strength and Limitations

All pregnant women in Norway were invited to participate in the MoBa study, but the attendance
rate was only 38.5%, rendering the cohort not representative of the Norwegian population of
pregnant women. The women who participated in the MoBa study are reported to be older, more
frequently vitamin users, non-smokers, and primiparous [32]. Pre-pregnancy weight was self-reported
around pregnancy week 18, and as obesity is an important determinant of underreporting BMI [33],
self-reporting without verification is a limitation of the study.
The samples were stored at −80 ◦ C before analyzing. The stability of plasma metabolites according
to sample handling and storage conditions has been validated and the biomarkers investigated do not
change significantly during storage at −80 ◦ C [34].
C-reactive protein, CRP, an acute phase reactant and a commonly used inflammation marker,
might have added additional information about low grade inflammation, but was not analyzed, which
is a limitation of the study.

4.3. Interpretation

4.3.1. Effects of Pre-Pregnancy BMI on Gestational Weight Gain and Birth Weight
In a former study of the MoBa cohort including approximately 58,000 mothers, the majority (65%)
reported a normal pre-pregnancy BMI (18.5–24.9), whereas 2.9% were underweight (<18.5), and 2.6%
were obese (≥35) [35], which is in accordance with the findings in the present substudy.
We observed a significant inverse relation between pre-pregnancy BMI and gestational weight
gain up to pregnancy week 18 and throughout pregnancy, and despite this, a higher pre-pregnancy
BMI was associated with a higher birth weight, which has been reported previously [35].

4.3.2. Pregnancy Related Changes in Biochemical Parameters

Physiological changes in pregnancy, including hemodilution, altered renal function, hormonal
status, and binding-protein concentrations, affect plasma B vitamin levels and the relation to their
functional metabolic markers [36,37]. Folate, cobalamin, and PLP tend to decrease, whereas riboflavin
is reported to show only minor changes during pregnancy [38–42]. tHcy, a marker of folate and
cobalamin deficiency [43], is 30%–60% lower in pregnant compared to nonpregnant women, with the
lowest levels seen in the second trimester [44]. MMA, a marker of cobalamin deficiency [45], is reported
to be reduced during the first two trimesters, thereafter gradually increasing [46]. We observed a strong
Nutrients 2016, 8, 776 11 of 15

inverse relation between folate and tHcy, and a weaker relation between cobalamin and MMA, as
observed by others [36,47,48].
The ratio HK/XA has been shown to have a strong negative correlation with plasma levels of
PLP, the active form of vitamin B6, and has been proposed as a potential markers of functional vitamin
B6 status [27]. The inverse relation between PLP and HK/XA has not been previously documented in
pregnant women, but we observed lower PLP and higher HK/XA ratios with increasing pre-pregnancy
BMI, indicating a functional vitamin B6 deficiency, particularly in women with a pre-pregnancy
BMI ≥30.
Extensive changes in the maternal immune system are necessary for maintaining a normal
pregnancy [49] and both neopterin levels and KTR are reported to increase during pregnancy [50]
Neopterin levels correlate with gestational age (r = 0.28, p = 0.001) and are reported to be higher in
pregnancies complicated with preterm birth and preeclampsia [51–53].

4.3.3. Maternal Predictors for B Vitamin Status

An inverse relation between pre-pregnancy BMI and folate, vitamin D, carotenoids has formerly
been reported [10,11,13]. Our results have expanded this knowledge by reporting also lower levels of
PLP (vitamin B6), riboflavin (vitamin B2), and cobalamin (vitamin B12) with higher pre-pregnancy BMI.
Pre-pregnancy BMI, use of multisupplements, parity, and maternal age were all related to
B vitamin status. We observed a slightly U-shaped relation between pre-pregnancy BMI and B vitamin
levels in pregnancy week 18, with the best status seen in women with a normal pre-pregnancy
BMI (18.5–24.9), slightly lower in underweight women (BMI < 18.5) and lowest in obese women
(BMI ≥35.0). The strongest maternal determinant for B vitamin status was use of multisupplements,
and the percentage of users was higher in normal weight compared to obese women, which might
explain the inverse relation between pre-pregnancy BMI and vitamin status. Use of micronutrient
supplements during pregnancy has been associated with better socioeconomic status [48], known
to be related to a better diet, maternal health, and pregnancy outcome [54]. Multiparity (≥3) was
associated with an increased risk of folate deficiency, while maternal age >25 years was associated
with a better folate status. In the MoBa cohort, the 10% who had used supplements regularly from one
month before pregnancy throughout the first trimester (as recommended) were more likely to be older,
married, nonsmokers, with a higher income and lower parity [54]. Compared to women with a lower
pre-pregnancy BMI, obese women (BMI ≥ 35) had not increased their weight in pregnancy week 18,
when the blood samples were collected, which might contribute to their lower micronutrient status.

4.3.4. The Complex Relation between Pre-pregnancy BMI, Inflammation, and Vitamin Levels
We observed a linear relation between pre-pregnancy BMI and the markers of cellular immune
activation, neopterin and KTR, as previously reported in non-pregnant adults [16]. Inflammation is
reported to be associated with lower plasma levels of several micronutrients, particularly vitamin
B6 and vitamin D [18–20], but the magnitude of effect varies between different micronutrients and
individuals [18,55,56]. The lower PLP levels associated with inflammation have been proposed to be
due to increased PLP degradation [57] and to PLP redistribution from plasma to tissues [58], and PLP
have been suggested to reflect interferon-γ-mediated immune activation [59].
In this study, neopterin was inversely related to PLP and positively to HK/XA, but not to any
other vitamin, in both multiple linear and logistic regression models, which included pre-pregnancy
BMI and other maternal factors. The increase in the functional marker, HK/XA, suggests that low PLP
does not merely refelect altered distribution, but that inflammation may be an independent risk factor
for lower vitamin B6 status.

5. Conclusions
A high pre-pregnancy BMI is associated with lower levels of folate, cobalamin, PLP, and riboflavin;
and increased levels of the cellular inflammation markers, neopterin, and KTR. As an optimal
Nutrients 2016, 8, 776 12 of 15

micronutrient status is vital for normal fetal development, the observed lower B vitamin levels
may contribute to the negative pregnancy outcomes associated with maternal obesity. Assessment
of B vitamin status should be encouraged in women with a high pre-pregnancy BMI when planning
a pregnancy.

Acknowledgments: We are grateful to all the families in Norway who took part in this cohort study.
The Norwegian Mother and Child Cohort Study is supported by the Norwegian Ministry of Health and the
Ministry of Education and Research, NIH/NIEHS (contract no. NO-ES-75558), NIH/NINDS (grant no.1 UO1 NS
047537-01), and the Norwegian Research Council/FUGE (grant no. 151918/S10).
Author Contributions: (1) Designed research: A.-L.B.-M., P.M.U., P.M., C.S., and S.E.V.; (2) Conducted research:
C.R., P.M., and C.S.; (3) Analyzed data: A.-L.B.-M., A.U., R.M.N., and Ø.M.; (4) Wrote the paper: A.-L.B.-M.,
and P.M.U.; (5) Primary responsibility for final content: A.-L.B.-M.; (5) Primary responsibility for final content:
A.-L.B.-M.; (6) All authors have read and approved the final version of the manuscript: A.-L.B.-M., A.U., Ø.M.,
R.M.N., C.R., P.M., C.S., S.E.V., T.R.-K., and P.M.U.
Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations
MoBa: the Norwegian Mother and Child Cohort; tHcy: total homocysteine; MMA: methylmalonic acid;
HK: 3-hydroxykynurenine; XA: xanthurenic acid; PLP: pyridoxal 5’-phosphate; KTR: kynurenine/tryptophan
ratio; IDO: indoleamine-2,3-dioxygenase.

References
1. Swinburn, B.A.; Sacks, G.; Hall, K.D.; McPherson, K.; Finegood, D.T.; Moodie, M.L.; Gortmaker, S.L.
The global obesity pandemic: Shaped by global drivers and local environments. Lancet 2011, 378, 804–814.
[CrossRef]
2. Catalano, P.M.; Ehrenberg, H.M. The short- and long-term implications of maternal obesity on the mother
and her offspring. Br. J. Obstet. Gynaecol. 2006, 113, 1126–1133. [CrossRef] [PubMed]
3. Stothard, K.J.; Tennant, P.W.; Bell, R.; Rankin, J. Maternal overweight and obesity and the risk of congenital
anomalies: A systematic review and meta-analysis. J. Am. Med. Assoc. 2009, 301, 636–650. [CrossRef]
[PubMed]
4. Boney, C.M.; Verma, A.; Tucker, R.; Vohr, B.R. Metabolic syndrome in childhood: Association with birth
weight, maternal obesity, and gestational diabetes mellitus. Pediatrics 2005, 115, e290–e296. [CrossRef]
[PubMed]
5. Kimmons, J.E.; Blanck, H.M.; Tohill, B.C.; Zhang, J.; Khan, L.K. Associations between body mass index and
the prevalence of low micronutrient levels among US adults. MedGenMed 2006, 8, 59. [PubMed]
6. Mojtabai, R. Body mass index and serum folate in childbearing age women. Eur. J. Epidemiol. 2004, 19,
1029–1036. [CrossRef] [PubMed]
7. Samuel, L.; Borrell, L.N. The effect of body mass index on optimal vitamin D status in U.S. adults:
The National Health and Nutrition Examination Survey 2001–2006. Ann. Epidemiol. 2013, 23, 409–414.
[CrossRef] [PubMed]
8. Nead, K.G.; Halterman, J.S.; Kaczorowski, J.M.; Auinger, P.; Weitzman, M. Overweight children and
adolescents: A risk group for iron deficiency. Pediatrics 2004, 114, 104–108. [CrossRef] [PubMed]
9. Strauss, R.S. Comparison of serum concentrations of alpha-tocopherol and beta-carotene in a cross-sectional
sample of obese and nonobese children (NHANES III). National Health and Nutrition Examination Survey.
J. Pediatr. 1999, 134, 160–165. [CrossRef]
10. Bodnar, L.M.; Catov, J.M.; Roberts, J.M.; Simhan, H.N. Pre-pregnancy obesity predicts poor vitamin D status
in mothers and their neonates. J. Nutr. 2007, 137, 2437–2442. [PubMed]
11. Kim, H.; Hwang, J.Y.; Kim, K.N.; Ha, E.H.; Park, H.; Ha, M.; Lee, K.Y.; Hong, Y.C.; Tamura, T.; Chang, N.
Relationship between body-mass index and serum folate concentrations in pregnant women. Eur. J.
Clin. Nutr. 2012, 66, 136–138. [CrossRef] [PubMed]
12. Tamura, T.; Goldenberg, R.L.; Johnston, K.E.; Chapman, V.R. Relationship between pre-pregnancy BMI and
plasma zinc concentrations in early pregnancy. Br. J. Nutr. 2004, 91, 773–777. [CrossRef] [PubMed]
Nutrients 2016, 8, 776 13 of 15

13. Tomedi, L.E.; Chang, C.C.; Newby, P.K.; Evans, R.W.; Luther, J.F.; Wisner, K.L.; Bodnar, L.M. Pre-pregnancy
obesity and maternal nutritional biomarker status during pregnancy: A factor analysis. Public Health Nutr.
2013, 16, 1414–1418. [CrossRef] [PubMed]
14. Allen, L.H. Multiple micronutrients in pregnancy and lactation: An overview. Am. J. Clin. Nutr. 2005, 81,
1206S–1212S. [PubMed]
15. Gregor, M.F.; Hotamisligil, G.S. Inflammatory mechanisms in obesity. Ann. Rev. Immunol. 2011, 29, 415–445.
[CrossRef] [PubMed]
16. Theofylaktopoulou, D.; Midttun, O.; Ulvik, A.; Ueland, P.M.; Tell, G.S.; Vollset, S.E.; Nygård, O.; Eussen, S.J.
A community-based study on determinants of circulating markers of cellular immune activation and
kynurenines: The Hordaland Health Study. Clin. Exp. Immunol. 2013, 173, 121–130. [CrossRef] [PubMed]
17. Visser, M.; Bouter, L.M.; McQuillan, G.M.; Wener, M.H.; Harris, T.B. Elevated C-reactive protein levels in
overweight and obese adults. J. Am. Med. Assoc. 1999, 282, 2131–2135. [CrossRef]
18. Duncan, A.; Talwar, D.; McMillan, D.C.; Stefanowicz, F.; O’Reilly, D.S. Quantitative data on the magnitude of
the systemic inflammatory response and its effect on micronutrient status based on plasma measurements.
Am. J. Clin. Nutr. 2012, 95, 64–71. [CrossRef] [PubMed]
19. Gori, A.M.; Sofi, F.; Corsi, A.M.; Gazzini, A.; Sestini, I.; Lauretani, F.; Bandinelli, S.; Gensini, G.F.; Ferrucci, L.;
Abbate, R. Predictors of vitamin B6 and folate concentrations in older persons: The InCHIANTI study.
Clin. Chem. 2006, 52, 1318–1324. [CrossRef] [PubMed]
20. Reyman, M.; Verrijn Stuart, A.A.; van Summeren, M.; Rakhshandehroo, M.; Nuboer, R.; de Boer, F.K.;
van den Ham, H.J.; Kalkhoven, E.; Prakken, B.; Schipper, H.S. Vitamin D deficiency in childhood obesity is
associated with high levels of circulating inflammatory mediators, and low insulin sensitivity. Int. J. Obes.
2014. [CrossRef] [PubMed]
21. Magnus, P.; Irgens, L.M.; Haug, K.; Nystad, W.; Skjaerven, R.; Stoltenberg, C. Cohort profile: The Norwegian
Mother and Child Cohort Study (MoBa). Int. J. Epidemiol. 2006, 35, 1146–1150. [CrossRef] [PubMed]
22. Nilsen, R.M.; Vollset, S.E.; Monsen, A.L.; Ulvik, A.; Haugen, M.; Meltzer, H.M.; Magnus, P.; Ueland, P.M.
Infant birth size is not associated with maternal intake and status of folate during the second trimester in
Norwegian pregnant women. J. Nutr. 2010, 140, 572–579. [CrossRef] [PubMed]
23. O’Broin, S.; Kelleher, B. Microbiological assay on microtitre plates of folate in serum and red cells.
J. Clin. Pathol. 1992, 45, 344–347. [CrossRef] [PubMed]
24. Kelleher, B.P.; Broin, S.D. Microbiological assay for vitamin B12 performed in 96-well microtitre plates.
J. Clin. Pathol. 1991, 44, 592–595. [CrossRef] [PubMed]
25. Baril, L.; Carmel, R. Comparison of radioassay and microbiological assay for serum folate, with clinical
assessment of discrepant results. Clin. Chem. 1978, 24, 2192–2196. [PubMed]
26. Windelberg, A.; Arseth, O.; Kvalheim, G.; Ueland, P.M. Automated assay for the determination of
methylmalonic acid, total homocysteine, and related amino acids in human serum or plasma by means
of methylchloroformate derivatization and gas chromatography-mass spectrometry. Clin. Chem. 2005, 51,
2103–2109. [CrossRef] [PubMed]
27. Ulvik, A.; Theofylaktopoulou, D.; Midttun, O.; Nygard, O.; Eussen, S.J.; Ueland, P.M. Substrate product
ratios of enzymes in the kynurenine pathway measured in plasma as indicators of functional vitamin B-6
status. Am. J. Clin. Nutr. 2013, 98, 934–940. [CrossRef] [PubMed]
28. Fuchs, D.; Weiss, G.; Wachter, H. Neopterin, biochemistry and clinical use as a marker for cellular immune
reactions. Int. Arch. Allergy Immunol. 1993, 101, 1–6. [CrossRef] [PubMed]
29. Raitala, A.; Pertovaara, M.; Karjalainen, J.; Oja, S.S.; Hurme, M. Association of interferon-gamma
+874(T/A) single nucleotide polymorphism with the rate of tryptophan catabolism in healthy individuals.
Scand. J. Immunol. 2005, 61, 387–390. [CrossRef] [PubMed]
30. Midttun, O.; Hustad, S.; Ueland, P.M. Quantitative profiling of biomarkers related to B-vitamin status,
tryptophan metabolism and inflammation in human plasma by liquid chromatography/tandem mass
spectrometry. Rapid Commun. Mass Spectrom. 2009, 23, 1371–1379. [CrossRef] [PubMed]
31. R Development Core Team. A Language and Environment for Statistical Computing; R Foundation for Statistical
Computing: Vienna, Austria, 2006.
32. Nilsen, R.M.; Vollset, S.E.; Gjessing, H.K.; Skjaerven, R.; Melve, K.K.; Schreuder, P.; Alsaker, E.R.; Haug, K.;
Daltveit, A.K.; Magnus, P. Self-selection and bias in a large prospective pregnancy cohort in Norway.
Paediatr. Perinat. Epidemiol. 2009, 23, 597–608. [CrossRef] [PubMed]
Nutrients 2016, 8, 776 14 of 15

33. Visscher, T.L.; Viet, A.L.; Kroesbergen, I.H.; Seidell, J.C. Underreporting of BMI in adults and its effect on
obesity prevalence estimations in the period 1998 to 2001. Obesity 2006, 14, 2054–2063. [CrossRef] [PubMed]
34. Hustad, S.; Eussen, S.; Midttun, O.; Ulvik, A.; van de Kant, P.M.; Morkrid, L.; Gislefoss, R.; Ueland, P.M.
Kinetic modeling of storage effects on biomarkers related to B vitamin status and one-carbon metabolism.
Clin. Chem. 2012, 58, 402–410. [CrossRef] [PubMed]
35. Stamnes Koepp, U.M.; Frost Andersen, L.; Dahl-Joergensen, K.; Stigum, H.; Nass, O.; Nystad, W.
Maternal pre-pregnant body mass index, maternal weight change and offspring birthweight. Acta Obstet.
Gynecol. Scand. 2012, 91, 243–249. [CrossRef] [PubMed]
36. Metz, J.; McGrath, K.; Bennett, M.; Hyland, K.; Bottiglieri, T. Biochemical indices of vitamin B12 nutrition in
pregnant patients with subnormal serum vitamin B12 levels. Am. J. Hematol. 1995, 48, 251–255. [CrossRef]
[PubMed]
37. Bruinse, H.W.; van den Berg, H. Changes of some vitamin levels during and after normal pregnancy. Eur. J.
Obstet. Gynecol. Reprod. Biol. 1995, 61, 31–37. [CrossRef]
38. Pietrzik, K.F.; Thorand, B. Folate economy in pregnancy. Nutrition 1997, 13, 975–977. [CrossRef]
39. Milman, N.; Byg, K.E.; Bergholt, T.; Eriksen, L.; Hvas, A.M. Cobalamin status during normal pregnancy
and postpartum: A longitudinal study comprising 406 Danish women. Eur. J. Haematol. 2006, 76, 521–525.
[CrossRef] [PubMed]
40. Shibata, K.; Tachiki, A.; Mukaeda, K.; Fukuwatari, T.; Sasaki, S.; Jinno, Y. Changes in plasma pyridoxal
5’-phosphate concentration during pregnancy stages in Japanese women. J. Nutr. Sci. Vitaminol. 2013, 59,
343–346. [CrossRef] [PubMed]
41. Cikot, R.J.; Steegers-Theunissen, R.P.; Thomas, C.M.; de Boo, T.M.; Merkus, H.M.; Steegers, E.A. Longitudinal
vitamin and homocysteine levels in normal pregnancy. Br. J. Nutr. 2001, 85, 49–58. [CrossRef] [PubMed]
42. Cunningham, P.; McDermott, L. Long chain PUFA transport in human term placenta. J. Nutr. 2009, 139,
636–639. [CrossRef] [PubMed]
43. Ueland, P.M.; Refsum, H.; Schneede, J. Determinants of plasma homocysteine. In Homocysteine and Vascular
Disease; Robinson, K., Ed.; Kluwer Academic Publishers: Dordrecht, The Netherlands, 2000; pp. 59–84.
44. Velzing-Aarts, F.V.; Holm, P.I.; Fokkema, M.R.; van der Dijs, F.P.; Ueland, P.M.; Muskiet, F.A. Plasma choline
and betaine and their relation to plasma homocysteine in normal pregnancy. Am. J. Clin. Nutr. 2005, 81,
1383–1389. [PubMed]
45. Savage, D.G.; Lindenbaum, J.; Stabler, S.P.; Allen, R.H. Sensitivity of serum methylmalonic acid and total
homocysteine determinations for diagnosing cobalamin and folate deficiencies. Am. J. Med. 1994, 96, 239–246.
[CrossRef]
46. Greibe, E.; Andreasen, B.H.; Lildballe, D.L.; Morkbak, A.L.; Hvas, A.M.; Nexo, E. Uptake of cobalamin and
markers of cobalamin status: A longitudinal study of healthy pregnant women. Clin. Chem. Lab. Med. 2011,
49, 1877–1882. [CrossRef] [PubMed]
47. Murphy, M.M.; Molloy, A.M.; Ueland, P.M.; Fernandez-Ballart, J.D.; Schneede, J.; Arija, V.; Scott, J.M.
Longitudinal study of the effect of pregnancy on maternal and fetal cobalamin status in healthy women and
their offspring. J. Nutr. 2007, 137, 1863–1867. [PubMed]
48. Bjorke-Monsen, A.L.; Roth, C.; Magnus, P.; Midttun, O.; Nilsen, R.M.; Reichborn-Kjennerud, T.;
Stoltenberg, C.; Susser, E.; Vollset, S.E.; Ueland, P.M. Maternal B vitamin status in pregnancy week 18
according to reported use of folic acid supplements. Mol. Nutr. Food Res. 2013, 57, 645–652. [CrossRef]
[PubMed]
49. Marques, A.H.; Bjorke-Monsen, A.L.; Teixeira, A.L.; Silverman, M.N. Maternal stress, nutrition and physical
activity: Impact on immune function, CNS development and psychopathology. Brain Res. 2015. [CrossRef]
[PubMed]
50. Schrocksnadel, K.; Widner, B.; Bergant, A.; Neurauter, G.; Schrocksnadel, H.; Fuchs, D. Tryptophan
degradation during and after gestation. Adv. Exp. Med. Biol. 2003, 527, 77–83. [PubMed]
51. Fuith, L.C.; Fuchs, D.; Hausen, A.; Hetzel, H.; Reibnegger, G.; Werner, E.R.; Wachter, H. Neopterin, a marker
of cell-mediated immune activation in human pregnancy. Int. J. Fertil. 1991, 36, 372–375. [PubMed]
52. Von Versen-Hoeynck, F.M.; Hubel, C.A.; Gallaher, M.J.; Gammill, H.S.; Powers, R.W. Plasma levels of
inflammatory markers neopterin, sialic acid, and C-reactive protein in pregnancy and preeclampsia.
Am. J. Hypertens. 2009, 22, 687–692. [CrossRef] [PubMed]
Nutrients 2016, 8, 776 15 of 15

53. Navolan, D.B.; Vladareanu, S.; Lahdou, I.; Ciohat, I.; Kleist, C.; Grigoras, D.; Vladareanu, R.; Terness, P.;
Sas, I. Early pregnancy serum neopterin concentrations predict spontaneous preterm birth in asymptomatic
pregnant women. J. Perinat. Med. 2016. [CrossRef] [PubMed]
54. Nilsen, R.M.; Vollset, S.E.; Gjessing, H.K.; Magnus, P.; Meltzer, H.M.; Haugen, M.; Ueland, P.M. Patterns and
predictors of folic acid supplement use among pregnant women: The Norwegian Mother and Child Cohort
Study. Am. J. Clin. Nutr. 2006, 84, 1134–1141. [PubMed]
55. Theofylaktopoulou, D.; Ulvik, A.; Midttun, O.; Ueland, P.M.; Vollset, S.E.; Nygard, O.; Hustad, S.;
Tell, G.S.; Eussen, S.J. Vitamins B2 and B6 as determinants of kynurenines and related markers of
interferon-gamma-mediated immune activation in the community-based Hordaland Health Study. Br. J. Nutr.
2014, 112, 1065–1072. [CrossRef] [PubMed]
56. Lepski, S.; Brockmeyer, J. Impact of dietary factors and food processing on food allergy. Mol. Nutr. Food Res.
2013, 57, 145–152. [CrossRef] [PubMed]
57. Ulvik, A.; Midttun, O.; Pedersen, E.R.; Eussen, S.J.; Nygard, O.; Ueland, P.M. Evidence for increased
catabolism of vitamin B-6 during systemic inflammation. Am. J. Clin. Nutr. 2014, 100, 250–255. [CrossRef]
[PubMed]
58. Chiang, E.P.; Smith, D.E.; Selhub, J.; Dallal, G.; Wang, Y.C.; Roubenoff, R. Inflammation causes tissue-specific
depletion of vitamin B6. Arthritis Res. Ther. 2005, 7, R1254–R1262. [CrossRef] [PubMed]
59. Torsvik, I.K.; Ueland, P.M.; Markestad, T.; Midttun, O.; Monsen, A.L. Motor development related to duration
of exclusive breastfeeding, B vitamin status and B12 supplementation in infants with a birth weight between
2000–3000 g, results from a randomized intervention trial. BMC Pediatr. 2015, 15, 218. [CrossRef] [PubMed]

© 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).